Telomerase, the enzyme that synthesizes the ends of chromosomes, consists of a RNA subunit (TERC) that serves as a template for the reverse transcriptase component, TERT. In contrast to TERC, TERT is strictly regulated in human cells and is rate-limiting for telomerase activity. While TERT is robustly expressed during embryonic development and detectable at lower levels in adult stem cells it is turned off in most differentiated cells in humans. In the absence of TERT, human cells cannot synthesize their telomeres (?end-replication problem'), experience progressive telomere shortening and undergo cellular senescence, which presents an important barrier for tumor development. TERT re-expression prevents senescence and endows human cells with extended lifespan. Indeed, around 85% of all human cancers have solved this end-replication problem by re-expressing TERT and are telomerase positive, while the remaining tumors use a recombination-based mechanism, ALT (Alternative Lengthening of Telomeres), to stabilize their telomeres. ALT is employed by 10-15% of human tumors and is particularly characteristic for tumors of mesenchymal origin (osteosarcomas, soft tissue sarcomas) but is also utilized by other cancer types including glioblastoma multiforme, breast cancer among others. One major gap in our understanding of ALT is that we have little mechanistic insight into how cancer cells develop and maintain the ALT phenotype and, consequently, no therapeutics have been developed to disrupt this cancer maintaining mechanism. One major barrier for progress in understanding ALT is the lack no experimental model systems that would allow to dissect this pathway. Here we propose to establish, for the first time, a genetic models system that will position us to perform forward genetic screens to identify regulators of ALT. To establish this model system we have used a haploid cancer cell line, Hap1, that, owing to its unique feature of harboring a haploid chromosome content, has been successfully used for genome?wide screens. We have generate a conditional (floxed) TERT knock-in allele in this TERT (and telomerase) positive Hap1 cell line with the goal to delete TERT, rendering Hap1 telomerase negative and induce the development of ALT. To generate this Hap1 knock-in cell line we have designed TALENS and CRISPR/CAS 9 nucleases that can be successfully used to generate knock-in cell lines. Here we propose to firmly establish single-cell derived Hap1 knock-in cancer line in Aim 1 and in Aim 2 will delete TERT to determine the consequences of telomerase inactivation and development of ALT. These two Aims will establish, for the first time, a genetic tool that would allow us to pursue our long-term goal of performing genome-wide genetic screens to identify critical regulators of the ALT pathway. The identification of genes involved in ALT would potentially provide therapeutical targets.